i used 12% SDS-Page and used Pc3 cells which are treated however i still can see the signal in the control band(non-treated pc3) in the caspase-3 cleavage any suggestion how to remove this band?
Hi Lucia,
You probably have "basal" apoptosis in your population, which is quite normal, but if your treatment is supposed to increase this, then it does not work well.
Were your cells in exponential phase ? If too confluent, cells trigger apoptosis.
It seems that your cells reach the maximal apoptosis at 6h.
If you want to emphasize this point, then try a shorter exposure.
Regarding your loading control, the exposure is too long and saturated.
Good luck !
Hi
how were the cells the day you took the sample? Did they look okay or kind of dead? If your control is experiencing apoptosis then maybe is an issue of the culture. Can you try to do this westerns with another stock of cells? Maybe defeosting another vial different from that stock will help?
I agree the above two comments and further I think of the possibility that during the protein harvest, cells undergo apoptosis artificially by such as trypsin treatment, centrifugation, and sonication etc.
Why not add QVD (caspase inhibitor) to the control sample?
It must work!!!
Yes that will work but you need to bear in mind that if you do that your control will turn into a positive control, hence you will end up needing a negative control anyway to validate your experiment. Make sure you use fresh tips for every sample to avoid the possibility of a contamination, the samples don't flush to nearest wells when you load the gel...etc. I will suggest to try with new cells if you have a new batch
1. Ensure that your culture is not confluent and there is not a large amount of dead cells. You can use PI-DAPI or hoechst staining.
2. You can grow your culture in petri dishes or 4-6-well plates. Wash your culture 2-3 times in culture dishes before lysis and lyse them in the dishes to avoid caspase activation during tripsinisation and cell harvesting.
3. PC3 is cancer cell line that may have a constant level of cleaved caspase. See papers with WB of active caspase in PC3.
4. Use procaspase antibodies.
when i harvest the cell are 70% convluence and not a lot of dead cell in the medium.. however i didn't collect the supernatant(only the one that stcks on the plate) in the controll whereas the treated one i collect both supernatant and the one on the plate.
are using trypsin have anything to do with this to the control i mean? because normally i scrape using the rubber policeman
ps. how to shorter the exposure? did you mean the incubated time before the sample are collected?
-First, collecting plated cells in the control condition and plated+floating cells in other conditions makes your experiment invalid. You have to harvest cells exactly the same way in each condition. You don't need floating cells, so i would recommend you to wash your cells with pbs and scrap them, the usual way.
- Trypsin won't make your cells die, unless you let them for a long time
- When i meant "shorter the exposure", it is the time of exposure during revelation.
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